Oomycetes (water molds) such as Saprolegnia, Branchiomyces and Aphanomyces are responsible for devastating infections of fish in aquaculture, fish farms and hobby fish tanks.
Members of the genus Saprolegnia cause saprolegniosis, a disease that is characterized by visible white or grey patches of filamentous mycelium on the body or fins of freshwater fish. If untreated, infection with Saprolegnia species (spp.) leads to death by haemodilution. Saprolegnia spp. also infect fish eggs by adhesion to and penetration of the egg membrane. Saprolegnia spp. are considered opportunistic pathogens that are saprophytes. Infection frequently occurs during the winter, often resulting in large-scale “winter kill” epidemics. Conditions which promote proliferation of Saprolegnia spp. and their infectious zoospores include abrupt decreases in water temperatures and high density fish farming activities, and also appear to render fish vulnerable to infection due to increased physiological stress and immune system suppression.
Saprolegnia parasitica is one of the most economically damaging fish pathogens, causing losses of millions of dollars annually worldwide, particularly in the salmon and trout markets. In addition to being an opportunistic pathogen, some S. parasitica strains are highly virulent and cause primary infections.
Branchiomycosis is another infection occurring in fresh water fish, primarily in carp and eel. It is caused by Branchiomyces sanguinis and B. demigrans, with affected fish showing prominent gill necrosis and respiratory distress. The disease occurs most commonly in ponds with abundant organic matter, and high ammonia levels.
Parasitic infections are often concurrent with or accompany oomycete infections in commercial fish farming settings. Such parasitic infections include those caused by Ichthyophthirius multifilis, Trichodina spp., Dactylogyrus spp. and Gyrodactylus spp.
Oomycetes, including Saprolegnia spp. and Branchiomyces spp. are filamentous eukaryotic microorganisms which have many fungus-like characteristics, but are not true fungi. Like the true fungi, they feed on decaying matter and grow as branching filaments with non-septate hyphae. However, their cell wall is not composed of chitin (as in the true fungi) but is composed of a mixture of cellulosic compounds and other β-glucans. Further, oomycetes have several clearly defined developmental stages that are not found in fungi. Recent molecular studies have shown however, that closely related virulence components are shared between oomycetes and fungi.
Prior art methods for treatment of pathogenic oomycete infections, including saprolegniosis and branchiomycosis suffer from various disadvantages. The chemicals Diquat (a herbicide), benzalkonium chlorides, copper sulfate and potassium permanganate have all been disclosed to be useful for treatment of branchiomycosis. None of these however, are approved by the U.S. Food and Drug Administration for disease control in food fishes.
U.S. Pat. No. 6,160,023 discloses use of bronopol (2-bromo-nitropropane-1,3-diol) for treatment and prophylaxis of S. parasitica infections in fish, and for disinfecting equipment used in raising fish. This compound is mainly effective against infection present in fish eggs, but not that occurring in fish. In addition, it is relatively toxic to commercially important fish species.
Malachite green (4-[(4-dimethylaminophenyl)-phenyl-methyl]-N,N-dimethyl-aniline) was previously widely used to control saprolegniosis. While this organic dye is very efficient at killing S. parasitica, its use has been banned since 2002 around the world, due to its carcinogenic, teratogenic and toxicological properties. This has resulted in a dramatic increase of Saprolegnia infections in commercial settings. Therefore, there is an urgent need for novel alternative methods of management of saprolegniosis.
Methods are also known for controlling parasitic infections in fish. U.S. Pat. No. 5,464,837 and U.S. Pat. No. 5,188,832 disclose use of triazine dione compounds; U.S. Pat. No. 5,313,911 discloses use of hydrogen peroxide; U.S. Pat. No. 6,054,454 discloses use of oxadiazine derivatives; U.S. Pat. No. 6,982,285 discloses use of benzoylurea derivatives; U.S. Pat. No. 6,117,457 discloses use of peracetic acid; U.S. Pat. No. 5,593,678 discloses use of orthovanadate salts; U.S. Pat. No. 5,504,081 discloses use of nitromethylene derivatives. None of these methods are known to be useful for simultaneous control of oomycete and parasitic infections.
Stilbene derivatives, including 4,4′-bis-(1,3,5-triazinylamino)stilbene-2,2′-disulfonic acid derivatives, are known fluorescent brightening agents which bind to polysaccharides having β-glucosidic linkages, including cellulose and chitin. Such compounds show antifungal activity, presumably due to interaction with and disruption of chitin microfibrils which constitute a major structural element in the fungal cell wall (Roncero et al. (1985) J. Bacteriol. 163:1180-1185). Antifungal effects of fluorescent brighteners have been demonstrated in true fungi which contain chitin in their cell walls, including plant pathogenic fungi (Seppanen et al. (2004) Plant Cell. Rep. 22:584-593), human pathogenic fungi-like yeasts (Roncero et al. (1988) J. Bacteriol. 170:1950-54), dermatophytes (Inamori et al. (1985) Chem. Pharm. Bull. (Tokyo) 33:2904-9) and red alga (Belliveau et al. (1990) Stain Technol. 65:303-311), but not in aquatic molds which contain cellulose instead of chitin in their cell walls.
U.S. Pat. No. 4,723,034 discloses 2-vinyl stilbene derivatives useful as fungicides for plant protection and wood preservation.
U.S. Pat. No. 5,359,131 and U.S. Pat. No. 5,852,015 disclose stilbene derivatives having anti-viral effects.
U.S. Pat. No. 5,879,674 discloses methods of protecting plant crops from insect attack by using stilbene derivatives to induce epizootic viral infections.
U.S. Pat. No. 6,919,452 and US patent application publication No. US 2005/0230662 disclose 4,4′-bis(1,3,5-triazinylamino)stilbene-2,2′-disulfonic acid derivatives and their use as fluorescent brightening agents.
Nowhere in the prior art is it taught or suggested that stilbene derivatives may be used to treat aquatic mold infections.